U.S. patent application number 16/078763 was filed with the patent office on 2019-03-21 for generation of functional beta cells from human pluripotent stem cell-derived endocrine progenitors.
This patent application is currently assigned to Novo Nordisk A/S. The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Nicolaj Stroeyer Christophersen, Ulrik Doehn, Mattias Hansson.
Application Number | 20190085295 16/078763 |
Document ID | / |
Family ID | 55588039 |
Filed Date | 2019-03-21 |
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United States Patent
Application |
20190085295 |
Kind Code |
A1 |
Christophersen; Nicolaj Stroeyer ;
et al. |
March 21, 2019 |
Generation of Functional Beta Cells From Human Pluripotent Stem
Cell-Derived Endocrine Progenitors
Abstract
The present invention relates to generation of functional beta
cells from human pluripotent stem cell-derived endocrine
progenitors. The present invention also relates to functional beta
cells produced by said methods and uses of said beta cells.
Inventors: |
Christophersen; Nicolaj
Stroeyer; (Virum, DK) ; Doehn; Ulrik;
(Oelstykke, DK) ; Hansson; Mattias; (Malmoe,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
55588039 |
Appl. No.: |
16/078763 |
Filed: |
February 24, 2017 |
PCT Filed: |
February 24, 2017 |
PCT NO: |
PCT/EP2017/054390 |
371 Date: |
August 22, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0696 20130101;
C12N 2501/91 20130101; C12N 2501/845 20130101; C12N 2501/15
20130101; C12N 2501/72 20130101; C12N 5/0676 20130101; C12N 2501/01
20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; C12N 5/074 20060101 C12N005/074 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 24, 2016 |
EP |
16157181.5 |
Claims
1.-14. (canceled)
15. A method for generating functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors, comprising the
steps of (1) culturing the stem cell-derived endocrine progenitor
cells in a basal medium comprising a histone methyltransferase EZH2
inhibitor, a TGF-beta signaling pathway inhibitor, Heparin, and
Nicotinamide, to obtain INS+ and NKX6.1+ double positive immature
beta cells and (2) culturing the beta cells obtained in step (1)
with 12% KOSR and GABA, to obtain the functional mature beta
cells.
16. The method according to claim 15, wherein the histone
methyltransferase EZH2 inhibitor is 3-Deazaneplanocin A
(DZNep).
17. The method according to claim 15, wherein the TGF-beta
signaling pathway inhibitor is Alk5iII.
18. The method according to claim 15, wherein the medium in step
(1) further comprises one or more additional agents selected from
group consisting of a gamma-secretase inhibitor, a cAMP-elevating
agent, a thyroid hormone signaling pathway activator, and
combinations thereof.
19. The method according to claim 18, wherein the one or more
additional agents is a gamma-secretase inhibitor that is DAPT.
20. The method according to claim 18, wherein the one or more
additional agents is a cAMP-elevating agent that is dbcAMP.
21. The method according to claim 18, wherein the one or more
additional agents is a thyroid hormone signaling pathway activator
that is T3.
22. The method according to claim 18, wherein the one or more
additional agents is DAPT and T3.
23. The method according to claim 18, wherein the one or more
additional agents is DAPT and dbcAMP.
24. The method according to claim 15, wherein the histone
methyltransferase EZH2 inhibitor is 3-Deazaneplanocin A (DZNep),
wherein the TGF-beta signaling pathway inhibitor is Alk5iII, and
wherein the medium in step (1) further comprises one or more
additional agents selected from the group consisting of DAPT,
dbcAMP, and T3.
25. The method according to claim 24, wherein the one or more
additional agents is DAPT.
26. The method according to claim 24, wherein the one or more
additional agents is dbcAMP.
27. The method according to claim 24, wherein the one or more
additional agents is T3.
28. The method according to claim 24, wherein the one or more
additional agent is DAPT and T3.
29. The method according to claim 24, wherein the one or more
additional agent is DAPT and dbcAMP.
30. The method according to claim 18, wherein step (2) further
comprises culturing the beta cells obtained in step (1) with one or
more additional agents selected from the group consisting of
Alk5iII and T3.
31. The method according to claim 30, wherein the one or more
additional agents is Alk5iII.
32. The method according to claim 30, wherein the one or more
additional agents is T3.
33. The method according to claim 30, wherein the one or more
additional agents is Alk5iII and T3.
34. The method according to claim 24, wherein step (2) further
comprises culturing the beta cells obtained in step (1) with one or
more additional agents selected from the group consisting of
Alk5iII and T3.
35. The method according to claim 34, wherein the one or more
additional agents is Alk5iII.
36. The method according to claim 34, wherein the one or more
additional agents is T3.
37. The method according to claim 34, wherein the one or more
additional agents is Alk5iII and T3.
Description
TECHNICAL FIELD
[0001] The present invention relates to methods of generating
functional mature beta cells from human pluripotent stem cells
derived endocrine progenitors.
BACKGROUND
[0002] Islet cell transplantation has been used to treat type 1
diabetic patients showing superior glucose homeostasis compared
with insulin therapy but this therapy is limited by organ
donations. Human Pluripotent stem cells (hPSCs) such as human
embryonic stem cells (hESCs) can proliferate infinitively and
differentiate into many cell types, including beta cells (BCs) and
may address the shortage of donor islets. Protocols to
differentiate hPSC into definitive endoderm, (DE), pancreatic
endoderm (PE) cells and endocrine progenitors (EP) in vitro have
been provided in WO2012/175633, WO 2014/033322 and WO2015/028614
respectively. It is challenging to make glucose-responsive
insulin-secreting BCs in vitro from hPSCs. Most protocols result in
insulin-producing cells that fail to recapitulate the phenotype of
BCs as they also co-express other hormones such as glucagon and are
unresponsive to glucose stimulation.
[0003] Rezania, A. et al. "Reversal of diabetes with
insulin-producing cells derived in vitro from human pluripotent
stem cells" Nature Biotechnology 32, 1121-1133 (2014) and Pagliuca,
F. W. et al. "Generation of Functional Human Pancreatic b Cells In
Vitro" Cell 159(2), 428-439, Oct. 9, 2014, reported the in vitro
differentiation of hESCs into insulin-secreting cells. Using static
incubation studies, cells from both groups were sensitive to
glucose stimulation showing approximately 2-fold increase in
insulin output after glucose stimulation. This response varied
however qualitatively and quantitatively from that of primary adult
beta cells. As comparison, human islet stimulation index is
reported to be two to ten or higher (Shapiro, J. A. M. et al.
"Islet Transplantation in seven patients with type 1 diabetes
mellitus using a glucocorticoid-free immunosuppressive regimen" New
England Journal of Medicine 343, 230-238, July 27 (2000).
[0004] The reported stem cell-derived BCs also failed to display
insulin response to glucose in a dynamic cell perfusion assay and
are thus functionally immature relative to primary human BCs.
[0005] Efficient protocol for making functional mature BCs from
hPSC-derived endocrine progenitors that can respond to glucose in a
dynamic cell perfusion assay is not known. It is critical to
improve current protocols to generate fully functional mature BCs
for a more consistent cell product similar to human islets to
obtain a predictable outcome following transplantation as well as
for screening purposes in vitro.
SUMMARY
[0006] The present invention relates to improved methods for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors. The present invention also
relates to glucose responsive fully differentiated beta-cells. The
present invention further relates to functional mature beta cells
obtainable by the methods of the present invention. The present
invention further relates to medical use of said cells inter alia
in the treatment of type I diabetes. The present invention may also
solve further problems that will be apparent from the disclosure of
the exemplary embodiments.
BRIEF DESCRIPTION OF DRAWINGS
[0007] FIG. 1 shows the screening approach where undifferentiated
human embryonic stem cells (hESCs) were differentiated into
Definitive Endoderm (DE) and reseeded in T75 flasks, where the
cells were further differentiated into Pancreatic Endoderm (PE) and
Endocrine Progenitor (EP). The Beta cells (BC) step 1 screen was
started at the EP stage and continued for 4-7 days, and analysed by
qICC monitoring and/or flow cytometry of NKX6.1+/INS+/GCG- cell
number. BC step 2 screen was started at the end of BC step 1 screen
and continued for 3-7 day period in 3D suspension cultures by
dissociating to single cells at the end of BC step 1 and
re-aggregation to clusters on orbital shaker at 50 rpm. Cells were
analysed by static and/or dynamic GSIS, INS protein content, ICC,
and qPCR.
[0008] FIG. 2 shows effect of compounds on INS+/NKX6.1+/GCG-
expression at day 4 of BC step 1 measured by Flow cytometry
(FC).
[0009] FIG. 3 shows additive effect when combining hits during BC
step 1 on INS+/NKX6.1+ cell number.
[0010] FIG. 4 shows timing studies of BC step 1 method.
[0011] FIG. 5 shows effect of compounds added for 7 days during BC
step 2 method on static GSIS.
[0012] FIG. 6A shows presence of glucose responsive insulin
secreting cells at day 3 of BC step 2.
[0013] FIG. 6B shows presence of glucose responsive insulin
secreting cells at day 7 of BC step 2.
[0014] FIG. 7 shows functionality of hESC-derived beta cells
demonstrated by dose dependent glucose and sulfonylurea mediated
insulin release in a dynamic fashion
[0015] FIG. 8 shows robust protocol induced functional beta cells
from independent pluripotent cell lines.
[0016] FIG. 9 Beta cell specific genes expressed in stem
cell-derived beta cells at day 9 of BC step 2.
[0017] FIG. 10 shows enrichment of beta cell markers by sorting for
INS+/NKX6.+ cells
[0018] FIG. 11 shows diabetic mice transplanted with stem
cell-derived beta cells show rapid lowering of blood glucose and
reversal of diabetes
[0019] FIG. 12 shows intraperitoneal glucose tolerance test (IPGTT)
of transplanted cells
[0020] FIG. 13 shows stem cell derived beta cells protect against
hyperglycemia post-streptozotocin treatment
[0021] FIG. 14 shows high levels of circulating human C-peptide in
transplanted mice
DESCRIPTION
[0022] The inventors of the present invention have performed
extensive small-molecule screens and identified a novel and simple
two-step method that generates functional mature
[0023] Beta cells (BC) from the human pluripotent stem cell-derived
endocrine progenitor stage. The first step of the protocol (BC step
1) induces high fraction of INS+ and NKX6.1+ double positive cells
and only few GCG positive cells. The second step of the protocol
(BC step 2) generates functional mature BC that respond strongly to
repeated glucose challenges in vitro. Importantly, the hPSC-derived
BC cells respond to repeated glucose +/-Exendin4 challenges in a
dynamic perfusion assay. The resulting functional mature BC also
respond to increased glucose levels in vivo 3 weeks after
transplantation to the kidney capsule of non-diabetic mice.
[0024] The inventors of the present invention have found that
gamma-Aminobutyric acid (GABA) administration in vivo following
cell transplantation can potentially potentiate functional effect
of transplanted BC. The resulting fully functional BC population
can be used as an in vitro-based BC product to study human BC
function, screening compounds for regulating insulin secretion,
insulin protein processing, insulin secretion and--mechanism, GSIS
studies, calcium influx signaling, autoimmune BC destruction, and
BC trans differentiation. Throughout this application terms method
or protocol or process may be used interchangeably.
Particular Embodiments
[0025] 1. A method for generation of functional mature beta cells
from human pluripotent stem cell-derived endocrine progenitors
comprising the steps of (1) culturing the stem cell-derived
endocrine progenitor cells in a medium comprising histone
methyltransferase EZH2 inhibitor, transforming growth factor beta
(TGF)-beta signaling pathway inhibitor, Heparin and Nicotinamide in
basal medium, to obtain INS+ and NKX6.1+ double positive immature
beta cells and (2) culturing the beta cells obtained in step (1)
with 12% KOSR and GABA , to obtain functional mature beta cells.
[0026] 2. The method for generation of functional mature beta cells
from human pluripotent stem cell-derived endocrine progenitors
according to embodiment 1, wherein histone methyltransferase EZH2
inhibitor is 3-Deazaneplanocin A (DZNep). [0027] 3. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 2,
wherein concentration of DZNep is below 1 .mu.M.
[0028] 4. The method for generation of functional mature beta cells
from human pluripotent stem cell-derived endocrine progenitors
according to embodiment 2, wherein concentration of DZNep is 1
.mu.M. [0029] 5. The method for generation of functional mature
beta cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 2, wherein concentration of
DZNep is in a range of 1-10 .mu.M. [0030] 6. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 2,
wherein concentration of DZNep is 10 .mu.M. [0031] 7. The method
for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 1, wherein transforming growth factor beta (TGF)-beta
signaling pathway inhibitor is Alk5iII. [0032] 8. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 7,
wherein concentration of Alk5iII is below 1 .mu.M. [0033] 9. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 7, wherein concentration of Alk5iII is 1 .mu.M. [0034]
10. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 7, wherein concentration of Alk5iII is in a range of
1-10 .mu.M. [0035] 11. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to embodiment 7, wherein
concentration of Alk5iII is 10 .mu.M. [0036] 12. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 1,
wherein concentration of Heparin is below 1 .mu.g/ml. [0037] 13.
The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 1, wherein concentration of Heparin is 1 .mu.g/ml.
[0038] 14. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 1, wherein concentration of
Heparin is in a range of 1-10 .mu.g/ml. [0039] 15. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 1,
wherein concentration of Heparin is 10 .mu.g/ml. [0040] 16. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 1, wherein the concentration of Nicotinamide is below 1
mM. [0041] 17. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 1, wherein the concentration of
Nicotinamide is 1 mM. [0042] 18. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 1,
wherein the concentration of Nicotinamide is in a range of 1-10 mM.
[0043] 19. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 1, wherein the concentration of
Nicotinamide is 10 mM. [0044] 20. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 1,
comprising the step of (1) culturing the stem cell-derived
endocrine progenitor cells in a medium comprising DZNep, Alk5iII,
Heparin and Nicotinamide. [0045] 21. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 1,
comprising the step of (1) culturing the stem cell-derived
endocrine progenitor cells in a medium comprising 1 .mu.M DZNep, 10
.mu.M Alk5iII, 10 .mu.g/ml Heparin and 10 mM Nicotinamide. [0046]
22. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 1, comprising step (1) in combination with one or
more additional agent. [0047] 23. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 22,
wherein the additional agent is selected from a group consisting of
gamma-secretase inhibitor, cAMP-elevating agent, thyroid hormone
signaling pathway activator and combinations thereof. [0048] 24.
The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 23, wherein the additional agent is gamma-secretase
inhibitor. [0049] 25. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to embodiment 24, wherein
gamma-secretase inhibitor is
N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethy-
l ester (DAPT). [0050] 26. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to embodiment 25, wherein the
concentration of DAPT is below 2.5 .mu.M. [0051] 27. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 25,
wherein the concentration of DAPT is 2.5 .mu.M. [0052] 28. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 25, wherein the concentration of DAPT is in a range of
2.5-10 .mu.M. [0053] 29. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to embodiment 25, wherein the
concentration of DAPT is 5 .mu.M. [0054] 30. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 25,
wherein the concentration of DAPT is 10 .mu.M. [0055] 31. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 23, wherein the additional agent is cAMP-elevating
agent. [0056] 32. The method for generation of functional mature
beta cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 31, wherein cAMP-elevating
agent is Dibutyryl-cAMP (dbcAMP). [0057] 33. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 32,
wherein the concentration of dbcAMP is below 250 .mu.M. [0058] 34.
The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 32, wherein the concentration of dbcAMP is 250 .mu.M.
[0059] 35. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 32, wherein the concentration
of dbcAMP is in a range of 250-500 .mu.M. [0060] 36. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 32,
wherein the concentration of dbcAMP is 500 .mu.M. [0061] 37. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 23, wherein the additional agent is thyroid hormone
signaling pathway activator. [0062] 38. The method for generation
of functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 37,
wherein thyroid hormone signaling pathway activator is T3. [0063]
39. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 38, wherein the concentration of T3 is below 1 .mu.M.
[0064] 40. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 38, wherein the concentration
of T3 is 1 .mu.M. [0065] 41. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 38,
wherein the concentration of T3 is in a range of 1-10 .mu.M. [0066]
42. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 38, wherein the concentration of T3 is 10 .mu.M.
[0067] 43. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 1, comprising the step of (1)
culturing the stem cell-derived endocrine progenitor cells in a
medium comprising DZNep, Alk5iII, Heparin and Nicotinamide in
combination with DAPT. [0068] 44. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 1,
comprising step of (1)culturing the stem cell-derived endocrine
progenitor cells in a medium comprising 1 .mu.M DZNep, 10 .mu.M
Alk5iII, 10 .mu.g/ml Heparin and 10 mM Nicotinamide in combination
with 2.5 .mu.M DAPT. [0069] 45. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 1,
comprising step of (1) culturing the stem cell-derived endocrine
progenitor cells in a medium comprising DZNep, Alk5iII, Heparin and
Nicotinamide in combination with dbcAMP. [0070] 46. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 1,
comprising step (1) culturing the stem cell-derived endocrine
progenitor cells in a medium comprising 1 .mu.M DZNep, 10 .mu.M
Alk5iII, 10 .mu.g/ml Heparin and 10 mM Nicotinamide in combination
with 250 .mu.M dbcAMP. [0071] 47. The method for generation of
functional beta cells from human pluripotent stem cell-derived
endocrine progenitors according to embodiment 23, wherein the
additional agent gamma-secretase inhibitor is in combination with
thyroid hormone signaling pathway activator. [0072] 48. The method
for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 47, wherein the additional agent DAPT is in combination
with T3. [0073] 49. The method for generation of functional mature
beta cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 48, wherein the concentration
of DAPT is 2.5 .mu.M and concentration of T3 is 1 .mu.M. [0074] 50.
The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 23, wherein the additional agent gamma-secretase
inhibitor is in combination with cAMP elevating agent. [0075] 51.
The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 50, wherein the additional agent is DAPT in
combination with dbcAMP. [0076] 52. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 51,
wherein the concentration of DAPT is 2.5 .mu.M and concentration of
dbcAMP is 250 .mu.M. [0077] 53. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 1,
wherein the stem cell-derived endocrine progenitor cells are
cultured in step (1) for 1-4 days.
[0078] 54. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 1, wherein the stem
cell-derived endocrine progenitor cells are cultured in step (1)
for 4 days. [0079] 55. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to embodiment 1, wherein the stem
cell-derived endocrine progenitor cells are cultured in step (1)
for 4-7 days. [0080] 56. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to any of one preceding
embodiments, wherein 10-60% INS+ and NKX6.1+ double positive
immature beta cells are obtained in step 1. [0081] 57. The method
for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
any one the preceding embodiments, wherein 20-50% INS+ and NKX6.1+
double positive immature beta cells are obtained in step (1).
[0082] 58. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to any of one preceding embodiments, wherein
25-45% INS+ and NKX6.1+ double positive immature beta cells are
obtained in step 1. [0083] 59. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to any of one
preceding embodiments, wherein 30-40% INS+ and NKX6.1+ double
positive immature beta cells are obtained in step 1. [0084] 60. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 1, comprising culturing the beta cells obtained in step
(1) with 12% KOSR and GABA, in combination with one or more
additional agent to obtain functional mature beta cells. [0085] 61.
The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 60, wherein the concentration of GABA is 50 .mu.M.
[0086] 62. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 60, wherein the concentration
of GABA is in a range of 50-250 .mu.M. [0087] 63. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 60,
wherein the concentration of GABA is 250 .mu.M. [0088] 61. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 60, wherein additional agent is TGF-beta signaling
pathway inhibitor. [0089] 62. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 61,
wherein TGF-beta signaling pathway inhibitor is Alk5iII. [0090] 63.
The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 62, wherein the concentration of Alk5iII is below 1
.mu.M. [0091] 64. The method for generation of functional mature
beta cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 62, wherein the concentration
of Alk5iII is 1 .mu.M. [0092] 65. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 62,
wherein the concentration of Alk5iII is in a range of 1-10 .mu.M.
[0093] 66. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 62, wherein the concentration
of Alk5iII is 10 .mu.M. [0094] 67. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 60,
wherein additional agent is thyroid hormone signaling pathway
activator. [0095] 68. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to embodiment 67, wherein thyroid
hormone signaling pathway activator is T3. [0096] 69. The method
for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 68, wherein the concentration of T3 is below 1 .mu.M.
[0097] 70. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 68, wherein the concentration
of T3 is 1 .mu.M. [0098] 71. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 68,
wherein the concentration of T3 is in a range of 1-10 .mu.M. [0099]
72. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 68, wherein the concentration of T3 is 10 .mu.M.
[0100] 73. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 60, wherein additional agent is
histone methyltransferase EZH2 inhibitor. [0101] 74. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 73,
wherein histone methyltransferase EZH2 inhibitor is DZNep. [0102]
75. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 74, wherein the concentration of DZNep is below 1
.mu.M. [0103] 76. The method for generation of functional mature
beta cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 74, wherein the concentration
of DZNep is in a range of 1-10 .mu.M. [0104] 77. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 74,
wherein the concentration of DZNep is 10 .mu.M. [0105] 78. The
method for generation of functional mature beta cells from human
pluripotent stem cell-derived endocrine progenitors according to
embodiment 60, wherein additional agent TGF-beta signaling pathway
inhibitor is in combination with thyroid hormone signaling pathway
activator and histone methyltransferase EZH2 inhibitor is DZNep.
[0106] 79. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 78, wherein additional agent
Alk5iII in combination with T3 and DZNep. [0107] 80. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 79,
wherein additional agent 10 .mu.M Alk5iII in combination with 1
.mu.M T3 and 1 .mu.M DZNep. [0108] 81. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 60,
wherein additional agent TGF-beta signaling pathway inhibitor is in
combination with thyroid hormone signaling pathway activator.
[0109] 82. The method for generation of functional mature beta
cells from human pluripotent stem cell-derived endocrine
progenitors according to embodiment 81, wherein additional agent
Alk5iII in combination with T3. [0110] 83. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to embodiment 82,
wherein 10 .mu.M Alk5iII is in combination with 1 .mu.M T3. [0111]
84. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 60, wherein additional agent TGF-beta signaling
pathway inhibitor is in combination with histone methyltransferase
EZH2 inhibitor is DZNep. [0112] 85. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to embodiment 84,
wherein additional agent Alk5iII in combination with DZNEP. [0113]
86. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to embodiment 85, wherein 10 .mu.M Alk5iII is in combination with
1.mu.DZNEP. [0114] 87. The method for generation of functional
mature beta cells from human pluripotent stem cell-derived
endocrine progenitors according to any of one preceding embodiments
60-86, wherein INS+ and NKX6.1+ double positive immature beta cells
obtained in step (1) are cultured in step (2) for 3-7 days. [0115]
88. The method for generation of functional mature beta cells from
human pluripotent stem cell-derived endocrine progenitors according
to any of one preceding embodiments 60-86, wherein INS+ and NKX6.1+
double positive immature beta cells obtained in step (1) are
cultured in step (2) for 7-11 days. [0116] 89. The method for
generation of functional mature beta cells from human pluripotent
stem cell-derived endocrine progenitors according to any of one
embodiments 60-86, wherein 10-60% functional mature beta cells are
obtained in step 2. [0117] 90. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to any of one
embodiments 60-86, wherein 20-50% functional mature beta cells are
obtained in step 2. [0118] 91. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to any of one
embodiments 60-86, wherein 25-45% functional mature beta cells are
obtained in step 2. [0119] 92. The method for generation of
functional mature beta cells from human pluripotent stem
cell-derived endocrine progenitors according to any of one
embodiments 60-86, wherein 30-40% functional mature beta cells are
obtained in step 2. [0120] 93. Functional mature beta cells
obtainable by method according to any one of embodiments 1-92.
[0121] 94. Functional mature beta cells obtained in embodiment 93
express MAFA, IAPP and G6PC2.
[0122] In one embodiment, the cells obtainable by the method
according to the invention are insulin producing cells, optionally
together with cells differentiated towards glucagon, somatostatin,
pancreatic polypeptide, and/or ghrelin producing cells. As used
herein, "insulin producing cells" refers to cells that produce and
store or secrete detectable amounts of insulin. "Insulin producing
cells" can be individual cells or collections of cells.
[0123] In another embodiment, the cell population comprising
pancreatic cells is obtained from a somatic cell population. In
some aspects the somatic cell population has been induced to
de-differentiate into an embryonic-like stem (ES, e.g., a
pluripotent) cell. Such de-differentiated cells are also termed
induced pluripotent stem cells (iPSC).
[0124] In another embodiment, the cell population comprising
pancreatic cells is obtained from embryonic stem (ES, e.g.,
pluripotent) cells. In some aspects the cell population comprising
pancreatic cells is pluripotent cells such as ES like-cells.
[0125] In another embodiment, the cell population comprising
pancreatic cells is embryonic differentiated stem (ES or
pluripotent) cells. Differentiation takes place in embryoid bodies
and/or in monolayer cell cultures or a combination thereof.
[0126] In another embodiment, the cell population is a population
of stem cells. In some aspects the cell population is a population
of stem cells differentiated to the pancreatic endocrine
lineage.
[0127] Stem cells are undifferentiated cells defined by their
ability at the single cell level to both self-renew and
differentiate to produce progeny cells, including self-renewing
progenitors, non-renewing progenitors, and terminally
differentiated cells. Stem cells are also characterized by their
ability to differentiate in vitro into functional cells of various
cell lineages from multiple germ layers (endoderm, mesoderm and
ectoderm), as well as to give rise to tissues of multiple germ
layers following transplantation and to contribute substantially to
most, if not all, tissues following injection into blastocysts.
[0128] Stem cells are classified by their developmental potential
as: (1) totipotent, meaning able to give rise to all embryonic and
extraembryonic cell types; (2) pluripotent, meaning able to give
rise to all embryonic cell types; (3) multi-potent, meaning able to
give rise to a subset of cell lineages, but all within a particular
tissue, organ, or physiological system (for example, hematopoietic
stem cells (HSC) can produce progeny that include HSC
(self-renewal), blood cell restricted oligopotent progenitors and
all cell types and elements (e.g., platelets) that are normal
components of the blood); (4) oligopotent, meaning able to give
rise to a more restricted subset of cell lineages than multi-potent
stem cells; and (5) unipotent, meaning able to give rise to a
single cell lineage (e.g., spermatogenic stem cells).
[0129] As used herein "differentiate" or "differentiation" refers
to a process where cells progress from an undifferentiated state to
a differentiated state, from an immature state to a less immature
state or from an immature state to a mature state. For example,
early undifferentiated embryonic pancreatic cells are able to
proliferate and express characteristics markers, like PDX1, NKX6.1,
and PTF1a. Mature or differentiated pancreatic cells do not
proliferate and do secrete high levels of pancreatic endocrine
hormones or digestive enzymes. E.g., fully differentiated beta
cells secrete insulin at high levels in response to glucose.
Changes in cell interaction and maturation occur as cells lose
markers of undifferentiated cells or gain markers of differentiated
cells. Loss or gain of a single marker can indicate that a cell has
"matured or fully differentiated." The term "differentiation
factor" refers to a compound added to pancreatic cells to enhance
their differentiation to mature endocrine cells also containing
insulin producing beta cells. Exemplary differentiation factors
include hepatocyte growth factor, keratinocyte growth factor,
exendin-4, basic fibroblast growth factor, insulin-like growth
factor-1, nerve growth factor, epidermal growth factor
platelet-derived growth factor, and glucagon-like peptide 1. In
some aspects differentiation of the cells comprises culturing the
cells in a medium comprising one or more differentiation
factors.
[0130] As used herein, "human pluripotent stem cells" (hPSC) refers
to cells that may be derived from any source and that are capable,
under appropriate conditions, of producing human progeny of
different cell types that are derivatives of all of the 3 germinal
layers (endoderm, mesoderm, and ectoderm). hPSC may have the
ability to form a teratoma in 8-12 week old SCID mice and/or the
ability to form identifiable cells of all three germ layers in
tissue culture. Included in the definition of human pluripotent
stem cells are embryonic cells of various types including human
blastocyst derived stem (hBS) cells in 30 literature often denoted
as human embryonic stem (hES) cells, (see, e.g., Thomson et al.
(1998), Heins et al. (2004), as well as induced pluripotent stem
cells (see, e.g. Yu et al. (2007); Takahashi et al. (2007)). The
various methods and other embodiments described herein may require
or utilise hPSC from a variety of sources. For example, hPSC
suitable for use may be obtained from developing embryos.
Additionally or alternatively, suitable hPSC may be obtained from
established cell lines and/or human induced pluripotent stem (hiPS)
cells.
[0131] As used herein "hiPSC" refers to human induced pluripotent
stem cells.
[0132] As used herein, the term "blastocyst-derived stem cell" is
denoted BS cell, and the human form is termed "hBS cells". In
literature the cells are often referred to as embryonic stem cells,
and more specifically human embryonic stem cells (hESC). The
pluripotent stem cells used in the present invention can thus be
embryonic stem cells prepared from blastocysts, as described in
e.g. WO 03/055992 and WO 2007/042225, or be commercially available
hBS cells or cell lines. However, it is further envisaged that any
human pluripotent stem cell can be used in the present invention,
including differentiated adult cells which are reprogrammed to
pluripotent cells by e.g. the treating adult cells with certain
transcription factors, such as OCT4, SOX2, NANOG, and LIN28 as
disclosed in Yu, et al. (2007); Takahashi et al. (2007) and Yu et
al. (2009).
[0133] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law).
[0134] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way.
[0135] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0136] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit of the invention.
List of Abbreviations
[0137] AA: Activin A [0138] BC: Beta cells [0139] bFGF: basic
fibroblast growth factor (FGF2) [0140] D'Am: D'Amour protocol
(Kroon et al., 2008) [0141] DAPT:
N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethy-
l ester [0142] DE: definitive endoderm [0143] DZNep:
3-Deazaneplanocin A [0144] EP: Endocrine Progenitor [0145] FC: Flow
cytometry [0146] GABA: gamma-Aminobutyric acid [0147] GSIS: glucose
stimulated insulin secretion [0148] hESC: human embryonic stem
cells [0149] hIPSC: human induced pluripotent cells [0150] hPSC:
human pluripotent stem cells [0151] KOSR: knockout serum
replacement [0152] PE: Pancreatic Endoderm [0153] RNA: ribonucleic
acid [0154] PCR: polymerase chain reaction [0155] PS: primitive
streak
EXAMPLES
[0156] In general, the process of differentiating hPSCs to
functional mature beta cells goes through various stages. An
exemplary method for generating functional beta cells from hPSCs in
vitro is outlined in FIG. 1.
Example 1
Preparation of Endocrine Progenitor Cells
[0157] hESCs (SA121) were cultured in DEF media (Cellectis)
supplemented with 30 ng/mLbFGF (Peprotech #100-18B) and 10 ng/mL
noggin (Peprotech #120-10C).
[0158] For adherent cultures, the hESCs were differentiated into DE
in T75 flasks using a Chir99021 and ActivinA based protocol in
WO2012/175633. DE was trypsinized using Tryple Select (Invitrogen
#12563-029) and reseeded as single cells in RPM11640 supplemented
with 100 ng/ml ActivinA (Peprotech #120-14E), 2% B27 (Invitrogen
#17504-044) and 0.1% PEST (Gibco#15140) in T75 flasks at 200 K/cm2.
DE cells were allowed to attach and differentiated into PE using a
LDN, AM508 based protocol in WO 2014/033322 followed by a four day
EP protocol in WO2015/028614.
[0159] To produce large numbers of beta cells, a scalable
suspension-based culture system was utilized by differentiating
clusters of hESCs into DE in shaker flasks in Multitron Standard
incubators (Infors) as suspension cultures (1 million/ml) at 70 RPM
using a Chir99021 and ActivinA based protocol in WO2012/175633
without requirement of a reseeding step. DE cells were further
differentiated into PE using a LDN, AM508 based protocol in WO
2014/033322 with the following slight modification: LDN is not
added at PE day 4-10. Generation of PE was followed by a four day
EP protocol WO2015/028614.
Example 2
Screening for Factors that Induce INS+/NKX6.1+ Co-Expression During
BC Step 1
[0160] As a first step towards generating fully functional mature
beta cells, we screened for factors to generate maximal numbers of
immature INS+/NKX6.1+ cells (BC step 1 screen). BC step 1 screen
was initiated at the EP stage using library of kinase inhibitors,
epigenetic regulators, redox and bioactive lipids supplemented with
some literature based compounds (in total 650 compounds of
interests) added on top of RPM11640+2%B27+10 mM Nicotinamide.
[0161] Compounds were screened for their ability to induce INS+,
NKX6.1+ double positive immature BCs and few GCG positive cells
within a 7 days period. Media change was performed daily. Cells
were fixed at day 4 and day 7 of BC step 1 and analysed for INS
NKX6.1 and GCG expression using flow cytometry (see Table 1 and
FIG. 2). Briefly, cells were dispersed into single-cell suspension
by incubation with TrypLE Express at 37.degree. C. for 10 min.
Cells were resuspended in 4% paraformaldehyde, washed in PBS
followed by incubation with primary antibodies overnight and then
secondary antibodies for 1 hour. The differentiated hPSCs
co-expressed C-peptide+/NKX6-1+with few cells expressing the
.alpha.-cell hormone glucagon (FIG. 2). When quantified by flow
cytometry, 48% of the cells co-expressed C-peptide+/NKX6-1, more
than previously reported with stem cell-derived beta cells
(reference Melton, Kieffer).
TABLE-US-00001 TABLE 1 FC analysis of BC step 1 method at BC day 7
#1 INS+/NKX6.1+ 20.8% INS+/NKX6.1- 19.8% INS-/NKX6+ 16.4% INS/GCG
12.3% INS+/GCG- 25.6% INS-/GCG+ 2% #1: DZNEP 1 uM + Alk5i 10 uM +
10 ug/ml Heparin
[0162] Hits identified in a primary screen were then combined
individually and added on top of the BC step 1 medium (see Table 2
and FIG. 3). Timing of studies revealed that BC step 1 has an
optimal length of 4-7 days based on mRNA expression of Ins and Gcg
(see FIG. 4).
Table 2: Shows Hit Compounds in BC Step 1 Medium
TABLE-US-00002 [0163] Compound name Target Structure Concentration
DAPT Notch ##STR00001## 2.5 .mu.M ALK5iII TGF-.beta. RI Kinase
##STR00002## 1 .mu.M, 10 .mu.M DZNEP PRC complex? ##STR00003## 1
.mu.M, 10 .mu.M Heparin 10 .mu.g/ml dbcAMP Increased cAMP levels
##STR00004## 250 .mu.M, 500 .mu.M Nicotinamide ##STR00005## 10 mM
T3 Thyroid receptor ##STR00006## 1 .mu.M, 10 .mu.M ##STR00007##
Example 3
Generation of Glucose Sensing Insulin Secreting Beta Cells from BC
Step 1
[0164] The key functional feature of a fully functional mature beta
cell is its ability to perform glucose stimulated insulin secretion
(GSIS). We screened for factors in BC step 2 that could induce
functional beta cells from the immature INS+/NKX6.1+ cells from BC
step 1.
[0165] BC step 2 screen was performed in suspension cultures. For
adherent cultures, cells in T75 flasks were trypsinized at the end
of BC step 1 using Tryple Select and transferring cells into low
attachment 9 cm petri dishes in suspension with RPM11640 medium
(Gibco#61870) containing 12% KOSR (ThermoFisher#10828028) and 0.1%
PEST, Gibco#15140.
[0166] Effects of selected compounds were then tested for a 7 day
period for induction of glucose-responsive cells in a static GSIS
setup (see FIG. 5). Briefly, cell clusters were sampled and
incubated overnight in 2.8 mM glucose media to remove residual
insulin. Clusters were washed two times in Krebs buffer, incubated
in 2.8 mM Krebs buffer for 30 min, and supernatant collected. Then
clusters were incubated in 16 mM glucose Krebs buffer for 30 min,
and supernatant collected. This sequence was repeated. Finally,
clusters were incubated in Krebs buffer containing 2.8 mM glucose
for 30 min and then supernatant collected. Supernatant samples
containing secreted insulin were processed using Human Insulin
ELISA (Mercodia).
[0167] Hits identified in a primary screen were then combined
individually and added on top of the 12% KOSR medium to generate
the optimal 7-day BC step 2 protocol (see Table 3).
Table 3: Shows Hit Compounds in BC Step 2 Medium
TABLE-US-00003 [0168] Compound name Target Structure Concentration
T3 Thyroid receptors ##STR00008## 1 .mu.M, 10 .mu.M ALK5iII
TGF-.beta. RI Kinase ##STR00009## 1 .mu.M, 10 .mu.M dbcAMP cAMP
##STR00010## 250 .mu.M GABA GABA receptors ##STR00011## 50 .mu.M
KOSR 12% ##STR00012##
Example 4
Perfusion Assay to Assay Dynamic Human Insulin Secretion In
Vitro
[0169] Mature beta cells are functionally defined by their rapid
response to elevated glucose. Secretion of human insulin by beta
cells at the end of BC step 2 was measured as repeated responses to
20 mM glucose.+-.1 .mu.M exendin-4 or .+-.the anti-diabetic
sulfonylurea compound Tolbutamide within a perfusion system.
[0170] Briefly, groups of 300 hand-picked, clusters of hESC- or
hiPSC-derived cell clusters were suspended with beads (Bio-Rad
#150-4124) in plastic chambers of Biorep PERFUSION SYSTEM (Biorep
#PERI-4.2). Under temperature- and CO2-controlled conditions, the
cells were perfused at 0.5 ml min-1 with a Krebs buffer. Prior to
sample collection, cells were equilibrated under basal (2 mM
glucose) conditions for 1 h. During perfusion cells were exposed to
repeated challenges with 20 mM glucose .+-.1 .mu.M exendin-4 or
.+-.100 .mu.M Tolbutamide. At the end of perfusion, cells were
exposed to cAMP-elevating agents on top of 20 mM glucose. Insulin
secretion was measured by human insulin ELISA (Mercodia).
[0171] By perfusion analysis, our stem cell-derived beta cells
exhibited rapid and robust release of insulin with a 1.sup.st and
2.sup.nd phase of insulin secretion that was highly synchronized
with changes in glucose concentrations (see FIG. 6). The GLP-1
analog exendin-4 increased the level of insulin secretion in the
hPSC-derived beta cells. Importantly, presence of glucose
responsive insulin secreting cells was observed for at least 4 days
in vitro as measured at day 3 (FIG. 6A) and day 7 (FIG. 6B) of BC
step 2.
[0172] Another example of perifusion analysis of our stem
cell-derived beta cells at day 7 of BC step 2 demonstrated a
significant additive effect of the sulfonylurea tolbutamide on
insulin secretion (FIG. 7). Robustness of the protocol is
demonstrated by induction of functional beta cells from independent
pluripotent cell lines (see FIG. 8). These data demonstrate
collectively the superiority of the protocol for generating stem
cell-derived beta cells that display glucose-stimulated insulin
release dynamics measured by perifusion as compared to previous
reports (Rezania, 2014; Paglucia, 2014 and review Johnson-J, 2016
Diabetologia).
Example 5
Gene Expression Analysis Showed High Level of Similarities of Stem
Cell-Derived Beta Cells to Human Islet Material
[0173] Differentiated cell clusters at BC day 7 of step 2 or human
islets were collected and RNA was purified using the RNeasy kit
from Qiagen (Cat No./ID: 74134). The quality was assessed using the
RNA 6000 Nano Kit and the 2100 Bianalyser instrument (Agilent). 100
ng RNA was subjected to an nCounter assay according to instructions
from Nanostring Technology. FIG. 9 shows the expression profile of
beta cell associated genes from human islets and beta cells
generated from hiPSC and two different hESC lines. The gene
expression analysis showed that the stem cell-derived beta cells
had close molecular resemblance to human islets.
[0174] Additional gene expression analysis of the specific stem
cell-derived INS+/NKX6.1 + cells were performed by FACS cell
sorting. Prior to sorting on the BD FACSARIA fusion.TM. instrument,
cell clusters were dissociated and stained for the separation of
live and dead cells using a near IR dye (Thermo Scientific). After
fixation and permeabilisation the cells were stained using the
intracellular markers NKX6.1 and C-peptide. RNA was purified using
the RNeasy FFPE Kit (QIAGEN) and quality was assessed using the RNA
6000 Nano Kit and the 2100 Bianalyser instrument (Agilent). FIG. 10
shows enrichment of key beta cell maturity genes after cell sorting
for NKX6.1/CPEP double positive cells. Nanostring data was
normalized to the unsorted cell population.
Example 6
Stem Cell-Derived Beta Cells from Step 2 Function After
Transplantation
[0175] To evaluate functionality in vivo, stem cell-derived beta
cells from day 3-10 of BC step 2 were transplanted into a
streptozotocin-induced mouse model of diabetes. In short, diabetes
is induced in immunocompromised scid-beige mice (Taconic) using
Multiple Low Dose (5.times.70 mg/kg) Streptozotocin (STZ), the mice
are fasted 4 h prior to STZ dosing. The mice are monitored over the
following weeks with respect to blood glucose, body weight and
HbA1c. Diabetes is considered when blood glucose is consistently
above 16 mM.
[0176] In full anaesthesia and analgesia the diabetic mice are
transplanted with 5.times.10.sup.6 human embryonic stem cell
derived beta cells (unsorted population) under the kidney capsule.
The kidney is exposed trough a small incision through skin and
muscle of the left back side of the animal, a pouch between the
parenchyma of the kidney and the capsule is created were the cell
clusters are injected. The abdominal wall and the skin is closed
and the mouse is allowed to recover.
[0177] The function of the cells is monitored over the coming weeks
with respect to blood glucose, body weight, HbA1c and human
c-peptide/insulin secretion. Our stem cell-derived beta cells
resulted in rapid reversal of diabetes within the first two weeks
after transplantation (FIG. 11), more rapidly than previous reports
(Rezania, 2014). Importanly, all mice with less than 85% of BW
received daily injections with insulin, i.e. non-transplanted
diabetic control group. In vivo challenge of transplanted cells
with glucose demonstrated in vivo functionality of our stem
cell-derived beta cells with better glucose clearance than control
mice and increased level of circulating human c-peptide within 60
min of glucose injection (FIG. 12). In another diabetes model, 5
million differentiated cells were transplanted to the kidney
capsule of non-diabetic SCID/Beige mice. These mice were then
treated with streptozotocin 8 weeks after transplantation. FIG. 13
demonstrates that the pre-transplanted mice were protected from
hyperglycemia post-streptozotocin administration versus
non-transplanted control mice, whereas removal of the graft
resulted in rapid hyperglycemia in the mice (see FIG. 13). High
levels of circulating human c-peptide was measured in all
transplanted mice from the first data point and until end of study
(see FIG. 14).
* * * * *